Biotechnology in Agricultural Chemistry - ACS Publications - American

Chapter 17

Genetic Engineering of Bacterial Insecticides Brian B. Spear

Downloaded by CORNELL UNIV on May 9, 2017 | http://pubs.acs.org Publication Date: March 18, 1987 | doi: 10.1021/bk-1987-0334.ch017

Abbott Laboratories, Chicago, IL 60064

Bacterial insecticides, especially B a c i l l u s thuringiensis, have become important factors i n insect control programs because of t h e i r e f f i c a c y and safety. B. thuringiensis produces a toxic protein that assembles into a c r y s t a l . Depending on the B. thuringiensis s t r a i n , the toxin i s s p e c i f i c for lepidoptera, mosquitoes, or beetles. Genes for the toxin have been cloned from several B. thuringiensis strains, analysed i n d e t a i l , and manipulated by recombinant DNA techniques. The toxic regions of these c r y s t a l proteins have been i d e n t i f i e d and sequenced. E f f o r t s to improve the i n s e c t i c i d a l properties of the toxin protein concentrate on y i e l d improvement, expression i n alternate hosts such as plants, and protein sequence changes. Progress toward these goals has been good, but more knowledge of the insect-toxin interactions i s required for a major breakthrough.

B i o l o g i c a l pesticides are becoming recognized as an important factor i n crop and forest protection and i n insect vector c o n t r o l . These pesticides are natural, disease-causing microorganisms such as viruses, bacteria and fungi, that i n f e c t or intoxicate s p e c i f i c pest groups. B i o l o g i c a l pesticides have great b i o l o g i c a l d i v e r s i t y , but nonetheless share several c h a r a c t e r i s t i c s . F i r s t , they a f f e c t a narrow spectrum of pests, usually w i t h i n a single order or even family. Second, they are very safe, due i n large part to t h e i r narrow spectrum of a c t i v i t y . They do not cause diseases i n vertebrates, and usually have no e f f e c t on predator species. Third, they share c e r t a i n l i m i t a t i o n s . Most are slow acting compared to chemicals, and since many must be i n f e c t i v e i n order to control pests, formulations must provide long term 0097-6156/87/0334-0204$06.00/0 © 1987 American Chemical Society

LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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Genetic Engineering of Bacterial Insecticides

v i a b i l i t y . Also, the i n s e c t i c i d a l microorganisms or t h e i r t o x i c products are s e n s i t i v e to environmental factors such as u l t r a v i o l e t l i g h t , plant surface chemicals, heat, and d e s s i c a t i o n . The greatest successes i n microbial pesticides have come from uses of the i n s e c t i c i d a l b a c t e r i a , B a c i l l u s thuringiensis s t r a i n HD-1 subspecies kurstaki (or BTk), and B. thuringiensis serotype H-14 subspecies i s r a e l e n s i s (or B T i ) . BTk i s e f f e c t i v e against f o l i a g e feeding c a t e r p i l l a r s of which over 150 have been documented, with the most notable being the cabbage looper, tobacco hornworm, tobacco budworm, European corn borer, gypsy moth, and spruce budworm. BTi on the other hand i s r e l a t i v e l y non-toxic to lepidoptera, but i s very e f f e c t i v e against mosquitoes and b l a c k f l i e s . In both cases B. thuringiensis acts as a stomach poison with the combined action of a protein toxin and septicemia due to germination of spores and b a c t e r i a l p r o l i f e r a t i o n w i t h i n the insect. The t o x i c i t y , production, and use of B. t h u r i n g i e n s i s has been succinctly reviewed by Krieg and Miltenburger [1). Uses for which BTk i s an accepted i n s e c t i c i d e range widely and include f o r e s t r y , vegetables, corn, tobacco, ornamentals, f r u i t trees, and stored grains. Sales of BTk are i n the range of $15 to $25 m i l l i o n annually. The success of BTk i s based on a combination of e f f i c a c y and safety. For pests such as cabbage looper, gypsy moth, and tobacco budworm, excellent control can be acheived with l e s s than 10 grams of BTk spores and c r y s t a l s per acre. Control of European cornborer with a granular formulation meets or exceeds the performance of the chemical i n s e c t i c i d e a l t e r n a t i v e s . The safety of BTk i s not only b e n e f i c i a l environmentally, but also has p r a c t i c a l consequences. Unlike most other i n s e c t i c i d e s , BTk does not require special protective clothing, there i s no waiting period before re-entering the f i e l d , and i t may be applied up to the day of harvest. BTk i s non-toxic to bees, and, because i t does not harm predatory insects, i s i d e a l l y suited to integrated pest management programs. Furthermore, i t can be used for a e r i a l spraying of r e s i d e n t i a l areas for control of gypsy moth, without fear of harm to humans or pets. The acceptance of BTk as an i n s e c t i c i d e i s best i l l u s t r a t e d by i t s *use i n gypsy moth control programs i n the U.S. When introduced i n 1981, BT was applied to 22,000 acres of gypsy moth infected forest, about 6% of treated acres. In 1985 and 1986 over 900,000 acres were treated annually, accounting for more than 70% of a l l pesticides applied during that time. Because of the intense environmental concern surrounding gypsy moth programs, and the demonstrated e f f i c a c y of BTk formulations, i t i s a n t i c i p a t e d that BTk w i l l further displace chemical i n s e c t i c i d e s i n f o r e s t r y . BTi i s a l s o gaining inceased acceptance as a l a r v i c i d e f o r mosquitoes and b l a c k f l i e s . Like BTk, BTi i s highly s p e c i f i c . While c o n t r o l l i n g most mosquito and b l a c k f l y species, i t does not have appreciable t o x i c i t y to most other f l i e s , and under f i e l d conditions i s harmless to non-dipteran insects. This environmental safety, coupled with a lack of human t o x i c i t y , makes BTi an i d e a l control agent for these b i t i n g pests. B l a c k f l i e s


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i n f e s t f a s t moving streams which can be important as drinking water or f o r sport f i s h i n g . Therefore, chemicals with any suspected mammalian t o x i c i t y or carcinogenicity, or that could wipe out insect populations necessary as d i e t f o r game f i s h , would be unacceptible. BTi i s now being used i n large programs t o control b l a c k f l i e s i n the U.S. and Canada, and under World Health Organization auspices i n West A f r i c a . The combination of safety and e f f i c a c y also make BTi an a t t r a c t i v e material for use i n mosquito control. Most mosquito i n s e c t i c i d e s are used only near r e s i d e n t i a l areas, necessitating the use of harmless materials. In addition to being safe t o humans, BTi does not harm b e n e f i c i a l insects or crustaceans often found i n mosquito habitats. Because BTi i s so e f f e c t i v e on i t s targets (use rates are as low as 5g/Acre) i t i s anticipated that use of BTi w i l l soon be the predominant i n s e c t i c i d e f o r mosquito larva control. Other b a c t e r i a l i n s e c t i c i d e s e x i s t , but have not yet acheived the commercial success of BT and BTi. B a c i l l u s sphaericus produces a protein that i s t o x i c p r i m a r i l y to Culex and Anopheles mosquitoes, and has the p o t e n t i a l f o r long term residual c o n t r o l . Formulations of B. sphaericus are under development and are expected to be available for public health use i n 1987. B a c i l l u s p o p i l l i a e and B a c i l l u s lentimorbus both i n f e c t the larvae~ô~ï scarabaeid beetles, including Japanese beetle grubs, causing milky disease. Both of these species are registered with EPA as b i o r a t i o n a l i n s e c t i c i d e s . Unlike B. thuringiensis, both species are obligate parasites and can be raised only i n beetle larvae. U n t i l a means of host-independent growth and sporulation of these species i s developed, i t i s u n l i k e l y that they w i l l be used on a broad scale. The use of B. p o p i l l a e and B. lentimorbus f o r grub control i s reviewed by B u l l a e t a l . ( 2 ) . The Molecular Biology of B a c t e r i a l Insecticides The i n s e c t i c i d a l toxins of B. thuringiensis and B. sphaericus are proteins, referred to as 6-endotoxins. These proteins form c r y s t a l l i n e structures, such as that shown i n figure 1, during sporulation. Despite having the common features of c r y s t a l formation and selective insect t o x i c i t y , the 6-endotoxins show considerable d i v e r s i t y between d i f f e r e n t s t r a i n s . The lepidopteran s p e c i f i c s t r a i n s have endotoxin proteins i n the range of 130,000d t o 140,000d [ f o r review see(_3)]. For instance, B.t. HD-73 has a δ-endotoxin with molecular weight of 133,000 (4); B.t. sotto has a 6-endotoxin with molecular weight of 144,000, (5) axU B.t. HD-l has two 6-endotoxins with molecular weights 130,000 and 135,000 which are the products of separate genes (6, 7). The 130-140,OOOd 6-endotoxin i s not t o x i c i n i t s native form, but must be cleaved to a 60-65,000 fragment to be a c t i v e . This cleavage takes place i n the insect gut. A f t e r ingestion by a c a t e r p i l l a r , the proteinaceous c r y s t a l dissolves i n the a l k a l i n e gut j u i c e s . Digestion by g a s t r i c proteases then cleave the



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protoxin i n t o the 65,000d a c t i v e form (8). The combined requirements f o r an a l k a l i n e environment f o r c r y s t a l d i s s o l u t i o n and appropriate proteases f o r 6-endotoxin a c t i v a t i o n contribute t o the varying degree of t o x i c i t y of BT t o various lepidoptera. The 6-endotoxin of B.t. i s r a e l e n s i s (H-14) i s not as c l e a r l y understood as that of BTk. The parasporal c r y s t a l of BTi contains several proteins with molecular weights ranging from 26,000 t o over 100,000d (9). Several authors have reported that the 26,000d p r o t e i n has i n s e c t i c i d a l a c t i v i t y (10, 11, 12). However, other reports suggest that the t o x i n i s 66,000d, (13, 14). Because BTi has hemolytic and c y t o l y t i c a c t i v i t i e s i n addition t o i n s e c t i c i d a l a c t i v i t i e s (15) the p o s s i b i l i t y e x i s t s that multiple t o x i n proteins e x i s t i n the c r y s t a l . However, evidence from genetics (12) and analysis of i s o l a t e d proteins (16) tends t o rule out a major role of the 65,000d protein as the primary i n s e c t i c i d a l t o x i n . Unlike the lepidopteran-specific BTk t o x i n , the 28,000d BTi protein does not appear t o be derived from a higher molecular weight polypeptide (17). A t h i r d class of B. thuringiensis termed Pathotype C has recently been discovered. (18, 19). This pathotype, now represented by 2 subspecies, B.t. tenebrionis and B.t. san diego, has a c r y s t a l l i n e protein that i s t o x i c t o beetles, But not t o mosquitoes or lepidoptera. The c r y s t a l i n pathotype C has a rectangular structure and i s composed almost e n t i r e l y o f a 64,000d protein (19). No higher molecular weight precursors t o the 64,000d protein were detected. The i n s e c t i c i d a l proteins of the three major B.t. pathotypes are a l l d i f f e r e n t . None of the three cross-react immunologically with the others (20, 19). A sequence of 30 amino acids of the BTi t o x i n protein has no r e l a t i o n s h i p the the lepidopteran s p e c i f i c BTk t o x i n sequence derived from the nucleotide sequence of the gene (10). However, a gene that encodes a t o x i n p r o t e i n from B t i has been sequenced, and the derived protein has s u b s t a n t i a l homology with a short region of the 6-endotoxin p r o t e i n from B.t. HD-1 (21). However, i t shares no homology with the 26,000d p r o t e i n from BTi. Indeed, i t i s not c l e a r which of the BTi c r y s t a l proteins i t might correspond to, since t h i s gene encodes a 58,000d protein, which has not been reported as a constituent o f the BTi c r y s t a l . B. sphaericus i s another species that produces a proteinaceous c r y s t a l which i s toxic t o mosquitoes (22). In B. sphaericus, the c r y s t a l i s enclosed w i t h i n the exosporium and so i s d i r e c t l y associated with the spore, unlike most B. thuringiensis subspecies where the c r y s t a l and spore are separate. The B. sphaericus c r y s t a l i s made up of several proteins with molecular weights ranging from 43,000d to w e l l over 100,000d (23). Only proteins of 43,000d and 63,000d remained a f t e r s o l u b i l i z a t i o n at high pH, and of these two, only the 43,000d protein showed i n s e c t i c i d a l a c t i v i t y . I t i s probable that the 43,000d t o x i n protein was derived from a 110,000d precursor and that, i n the insect gut, the 43,000d protein i s cleaved t o a 40,000d product.


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Recent evidence indicates that c y t o l y t i c a c t i v i t y of the B. sphaericus toxin requires pre-treatment with mosquito g a s t r i c j u i c e s (24), suggesting that proteolysis i s required f o r t o x i n a c t i v a t i o n , as i s the case with the lepidopteran s p e c i f i c BTs. The B. sphaericus toxin protein has no homology with BT toxins by the c r i t e r i a of immunoreactivity (23) or DNA h y b r i d i z a t i o n (25). However, data from DNA or protein sequence comparisons w i l l be necessary for an examination of possible d i s t a n t r e l a t i o n s h i p s .


Genetic Engineering of B a c t e r i a l Insecticides The 6-endotoxin gene from BTk was f i r s t cloned by recombinant DNA techniques i n 1981 (26). Since then, the toxin genes have been cloned from a v a r i e t y of B.t. s t r a i n s and from B. sphaericus. A l i s t of these cloned genes i s i n Table I . The i s o l a t i o n of these genes enables both the analysis and manipulation necessary t o a l t e r or improve the i n s e c t i c i d a l proteins. A major target i n toxin gene analysis has been t o i d e n t i f y protein regions that provide the t o x i c i t y or insect s p e c i f i c i t y of the 6-endotoxins. To date the complete DNA sequence of t o x i n genes from 4 B.t. s t r a i n s have been reported (Table I ) . The structure of cloned genes and t h e i r regulatory elements have been w e l l described i n recent papers (27, 28). When the B.t. 6-endotoxin i s p r o t e o l y t i c a l l y activated i n the insect gut, i t i s cleaved to a primary product of approximately 60,000d. By analysis of proteins synthesized from experimentally truncated, cloned genes, i t has been shown that the active p o r t i o n of the 6-endotoxin i s a t the amino end of the p r o t e i n (29, A, 30, 31). Schnepf and Whiteley working with BTk found that removal o f the f i r s t 50 amino acids of the 6-endotoxin p r o t e i n eliminated t o x i c i t y , but that removal of only the f i r s t 10 d i d not. A t the carboxy-terminal end, truncation of the protein a t amino a c i d 603 eliminated t o x i c i t y whereas truncation a t amino a c i d 645 d i d not (29). Thus, the t o x i c moiety i s between amino acids 10 and 645. Others working with a gene from B.t. HD-73 were able t o truncate to amino acid 612 without l o s i n g t o x i c i t y (j4 ). Because the genes analysed by the two groups d i f f e r e d from each other i n nucleotide sequences, the results are not d i r e c t l y comparable. A comparison of the reported B.t. toxin genes indicates that sequences of genes have diverged from each other i n some regions more than i n other (28). Interestingly, these regions can be correlated with the t o x i c and non-toxic parts of the protein. The f i r s t t h i r d of each of the proteins (from the amino terminus) share v i r t u a l l y i d e n t i c a l sequences. However, the center t h i r d shows considerable divergence from sequence t o sequence with some regions of nearly complete non-homology. The carboxy-terminal t h i r d of the genes re-establish homology, although not as t i g h t l y as the amino terminus. The point at which the non-homology of the central sequence changes t o the homology of the carboxy-terminal t h i r d i s a t amino acids 600-610, coincident with the terminus of the t o x i c moeity. The evolutionary conservation of the



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F i g . 1. Electron micrograph of B. thuringiensis HD-1 showing c r y s t a l and spore.

Table I . Cloned Genes f o r B a c t e r i a l I n s e c t i c i d a l Proteins Serotype

Sequence Available

Reference

B.t. kurstaki HD 1

Yes

7, 21, 26, 34, 35

B.t. kurstaki HD 73

Yes

4, 6

B.t. b e r l i n e r 1715

No

B.t.

sotto

Yes

32 5, 36

B.t. aizawai

No

36

B.t. subtoxicus

No

36

B.t. san diego

No

19

B.t. thuringiensis HD 2

No

6

B.t. i s r a e l e n s i s IPS 78

No

17

B.t. i s r a e l e n s i s QNR60A

Yes

21

B.t. i s r a e l e n s i s IPS 82

Yes

11

B.t. i s r a e l e n s i s HD 567-1

No

38

B. sphaericus

No

39


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carboxy-terminal region of the 6-endotoxin suggests that i t must have b i o l o g i c a l function, but that function i s not d i r e c t l y related to t o x i c i t y and i s unknown. The information on the structure and t o x i c i t y of the 6-endotoxin genes has been useful i n designing experiments t o a l t e r the a c t i v i t y or function of these proteins. In general, the genetic engineering approaches to b a c t e r i a l i n s e c t i c i d e s f a l l i n t o three categories. The f i r s t i s to change the y i e l d or potency o f e x i s t i n g b a c t e r i a l toxins. Second i s to change the host organism that c a r r i e s and expresses the genes. And t h i r d i s to change the spectrum of t o x i c i t y , to a l t e r or increase the i n s e c t i c i d a l range. Because of the amount of information a v a i l a b l e on lepidoptera-specific BT 6-endotoxins, almost a l l of the published genetic engineering work has been on these proteins. Although the current e f f o r t s on BT genetic engineering are intense, the number of research reports i s small, p r i m a r i l y because most work i s now being done by commercially oriented organizations rather than universities. The 6-endotoxin of BT accounts for over 30% of the p r o t e i n o f the sporangium. Therefore, e f f o r t s to increase the o v e r a l l synthesis are u n l i k e l y to r e s u l t i n substantial y e i l d improvement. Nevertheless, current studies to elucidate the regulatory elements of 6-endotoxin genes may lead to useful production increases. The promoter regions from several B.t. genes have been analysed and a l l appear to have i d e n t i c a l sequences (4, 5, 27). Wong, Schnepf and Whiteley found that two promoters function i n B. thuringiensis, one e a r l y i n the stationary phase and one i n mid and l a t e stationary phase. A t h i r d promoter i s active f o r t u i t i v e l y i n E. c o l i . In B. s u b t i l i s , cloned 6-endotoxin genes can be expressed at high l e v e l (7) but the l o c a t i o n of the promoter has not been reported. In f a c t , the f o r t u i t o u s E. c o l i promoter has more homology to a consensus B. s u b t i l i s promoter than do e i t h e r of the B. thuringiensis promoters. The organization of promoter sequences i n Bt have been reviewed recently (28). Despite the work on the structure o f the 6-endotoxin gene promoters, no a l t e r e d genes with enhanced 6-endotoxin synthesis have been described. An a l t e r n a t i v e approach to increasing 6-endotoxin y i e l d i s t o reduce the time required for a B.t. fermentation cycle. Recently we demonstrated that a 6-endotoxin gene from BTk can d i r e c t the synthesis of c r y s t a l s i n Ek s u b t i l i s without a requirement f o r stationary phase o f growth (Figure 2) (7). This contrasts with r e s u l t s from a cloned gene from B.t. b e r l i n e r i n which the 6-endotoxin was synthesized only during sporulation (32). The cloned B.t. b e r l i n e r gene i s flanked by several thousand base p a i r s of B.t. DNA, including a region of substantial secondary structure near the promoter sequence (33). The BTk gene, on the other hand was constructed with very l i t t l e B.t. DNA adjacent t o the promoter (7) suggesting that upstream sequences may be involved i n sporulation-restricted expression. Expression of B.t. genes during vegetative growth might shorten the o v e r a l l time



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F i g . 2. Electron micrograph of B. s u b t i l i s which carry a B. thuringiensis HD-1 gene on a recombinant plasmid. A p r o t e i n crystal i s visible.



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required f o r maximum c r y s t a l production, or allow f o r continuous fermentation processes. Recently several groups have been successful i n t r a n s f e r r i n g B.t. 6-endotoxin genes into alternate host organisms to s u b s t a n t i a l l y change the means of application of the i n s e c t i c i d e . In a much publicized project, Monsanto s c i e n t i s t s have introduced a cloned B.t. 6-endotoxin gene into the corn root colonizing bacterium, Pseudomonas fluorescens (34). This, i n concept, can be used to d e l i v e r the i n s e c t i c i d a l c r y s t a l s beneath the surface of the s o i l i n areas where root feeding insects do the most damage. Unfortunately, because of r e s t r i c t i o n s to the use of such recombinant organisms i n the environment, the u t i l i t y of t h i s approach has not yet been demonstrated. A second use of Ps. fluorescens has been developed by a research group at Mycogen Corp. Ps. fluorescens w i l l express the B.t. 6-endotoxin at high l e v e l s when i t c a r r i e s the B.t. gene on a recombinant plasmid. Unlike B. thuringiensis however, Pseudomonas does not sporulate. The recombinant Pseudomonas can be k i l l e d during production, thus providing a source of recombinant but non-viable b a c t e r i a l i n s e c t i c i d e . Because of the n o n - v i a b i l i t y of the product, the Environmental Protection Agency has approved f i e l d tests f o r t h i s material. Mycogen s c i e n t i s t s believe that encapsulation of the B.t. c r y s t a l by Pseudomonas c e l l w a l l w i l l protect the 6-endotoxin from environmental factors. Performance data on t h i s material i n the f i e l d are not yet a v a i l a b l e . An e x c i t i n g new approach to the use of B.t. 6-endotoxin has been the introduction of B.t. genes into the genome of a plant. Groups at Agrigenetics Corp and Plant Genetic Systems have transferred B.t. 6-endotoxin genes into a plasmid of the i n f e c t i o u s bacterium Agrobacterium tumifaciens and used the Agrobacterium to introduce t h i s plasmid i n t o tobacco plants. The r e s u l t s indicate that not only do plant tissues synthesize 6-endotoxin, but the leaves are also t o x i c to the tobacco hornworm. As methods f o r introducing foreign genes i n t o plants become available f o r more species, t h i s could become a widespread method f o r crop protection. Improvement of b a c t e r i a l i n s e c t i c i d e s through protein engineering to a l t e r the spectrum of insects that can be c o n t r o l l e i s the major long term goal of many reseach programs. I t i s not a easy task. As y e t , no r e s u l t s have appeared i n e i t h e r the s c i e n t i f i c or popular l i t e r a t u r e . Some short-term approaches have been suggested, such as changing the size of the 6-endotoxin through gene truncation, or forming chimeras of genes from d i f f e r e n t subspecies to make combinations not found i n nature. As a model system, i t was recently shown that a gene made by fusion of part of the 6-endotoxin gene and part of a drug resistance gene w i l l express both a c t i v i t i e s (30). The more long range approach involves directed mutagenesis of 6-endotoxin genes. This w i l l result i n replacement of amino a c i d residues or additions or deletions to the t o x i c protein. The technology f o r mutagenesis at s p e c i f i c s i t e s w i t h i n a gene i s w e l l


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developed, and the a b i l i t y to a l t e r protein function through such procedures has been demonstrated. However, s i t e - d i r e c t e d mutagenesis of 6-endotoxin genes to make new i n s e c t i c i d e s i s a p a r t i c u l a r l y d i f f i c u l t task. The l i m i t e d knowledge of the p r o t e i n structure i n insect t o x i c i t y makes the exact i d e n t i f i c a t i o n of appropriate s i t e s for mutation impossible. As an a l t e r n a t i v e , random mutations can be made w i t h i n the 600 amino a c i d region known to be t o x i c . The problem here i s the high cost and r e l a t i v e imprecision of the assays needed to screen the mutant proteins f o r altered insecticidal a c t i v i t y . The greatest l i m i t a t i o n to advances i n genetic engineering of b a c t e r i a l i n s e c t i c i d e s i s knowledge. The techniques f o r gene i s o l a t i o n , analysis and manipulation are w e l l developed and i n wide use. However, we are generally ignorant of the b i o l o g i c a l properties of the b a c t e r i a l t o x i n proteins and t h e i r i n t e r a c t i o n s with i n s e c t s . The nature of target s i t e s w i t h i n the insect gut i s unknown. Data on the mode of action of ô-endotoxins i s diverse and c o n f l i c t i n g . Certain lepidopterans are more susceptible to some s t r a i n s of B.t. than to others, but the biochemical or molecular basis of t h i s s p e c i f i c i t y i s unknown. B a c t e r i a l i n s e c t i c i d e s are an a t t r a c t i v e a l t e r n a t i v e to conventional pesticides and are an excellent target for improvement by genetic engineering. With integrated e f f o r t s i n molecular biology and insect physiology we should see s i g n i f i c a n t developments i n b a c t e r i a l i n s e c t i c i d e s i n the next f i v e or ten years. References 1. 2.

3. 4. 5. 6.

7. 8. 9. 10. 11. 12.

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